Historically, the detection and identification of bacteria, mycobacteria, yeasts, and molds have relied primarily on their morphologic and phenotypic properties. This approach is imprecise and slow for many clinically significant microbes. For this reason, we have explored alternative methods, such as Sanger sequencing, pyrosequencing, and mass spectroscopy, for the detection and identification of selected organisms. Earlier studies from our laboratory demonstrated that Sanger sequencing and pyrosequencing were powerful tools for the definitive identification of bacteria including Nocardia and Mycobacterium, yeasts, and molds; however, the procedures are expensive and time-consuming. Studies in our laboratory have focused on alternative identification methods, specifically MALDI-TOF (matrix-assisted laser desorption-ionization time of flight mass spectrometry) for the identification of aerobic and anaerobic bacteria, mycobacteria, and yeasts. MALDI-TOF MS provides a reproducible spectral pattern based on the mass/charge (m/z) ratio of ionized proteins. Preparation of samples is rapid and inexpensive. Following a demonstration of excellent performance, MALDI-TOF MS has been selected as the method of choice of the NIH Microbiology Service for bacterial, mycobacterial and yeasts identification. Very recently, our laboratory evaluated this technology for the identification of molds. Well-characterized reference and clinical strains (identified morphologically and by genomic sequencing) were used to build a NIH mold database that was then challenged with clinical isolates. MALDI TOF MS performance for mold identification was comparable to genomic sequencing with a much faster turn around time and lower expense cost. Implementation of this approach as the routine identification method in the clinical mycology laboratory is underway.